1. Field of the Invention
The invention relates to a method, in which specimens are examined with a microscope. For the illumination of the specimen, spatially coherent light in at least one continuous or continuously tunable wavelength range is generated. One or more wavelengths or ranges of wavelengths of the illumination light are selected in dependence on the specimen and/or the specified method of examination. The specimen is illuminated with the selected wavelengths or wavelength ranges. The illumination light and the emission light coming from the specimen are separated into separate beam paths. The illumination light is radiated back by the specimen and is suppressed in the beam path before the detection, and the emission light is detected. Further, the invention relates also to microscopes with which a specimen is analyzed—in particular by means of such a method. The present invention is concerned with the problem of the use of light sources, which emit spatially coherent light with a broad wavelength range, in microscopy, especially in fluorescence microscopy with Laser Scanning Technology (LST), and with the problems found in the obtained images, which arise in the use of the invention.
2. Description of Related Art
Use of broadband emission or variable broadband light sources in laser microscopy is attracting increasing interest recently. Its advantage lies in the free selection of the wavelength, which enables flexible adjustment of the different wavelengths that are needed in the excitation of the dyes, with which the specimens to be examined are marked. Use of such sources, which emit broadband, spatially coherent light, is described, for instance in U.S. Pat. No. 6,611,643. In that publication, a microscope apparatus comprising a laser is disclosed, in which the radiation is coupled in the microstructures of a fiber, in which broadband light is generated. However, the details concerning how the microscope itself is to be embodied, enabling it to use the broadband light in a suitable manner for various methods of examination, are not provided in the patent.
Instead of generating broadband light with microstructured fibers, the light can also be generated by exploiting the special properties of doped glass fibers, which are easier to handle than the aforementioned microstructured fibers. Such methods are described, for instance, in the article of Tauser et al in “Optical Letters” vol. 29 on pages 516 to 518.
A broadband laser illuminator is also described in U.S. Pat. No. 6,796,699. Here as well, however—apart from a general reference to the possibility of adaptation of the illuminator—no concrete technical embodiment for the adaptation of such an illuminator is described in the context of microscopy. Similarly, in U.S. Pat. No. 6,888,674 B1, only the coupling with a source with spectral broadening in a laser scanning microscope, abbreviated as LSM in the following, is described in very general terms, whereby the use of acousto-optical filters (AOTF), acousto-optical deflectors (AOD), acousto-optical beam splitters or LCD attenuators is concisely described.
In U.S. Pat. No. 6,654,166, the illumination of an LSM with an illumination unit is described, in which spectral broadening is generated with a microstructured fiber and a special beam splitter. The beam splitter is thereby a central element in the LSM, which must be specially adapted to the issues related to imaging and manipulation. The beam splitter described in U.S. Pat. No. 6,654,166 is embodied as a polarization- and wavelength-dependent element, which can also be prepared with a broadband reflecting coating, for example, of silver or aluminum. One must note, however, that in this embodiment there is the disadvantage that considerable losses result in the detection beam path. In particular, in fluorescence microscopy, these losses can lead to the failure of the method.
In combination with a broadband emitting or a variably tunable broadband laser, an accordingly suitable, flexible main dichroic beam splitter is required, which separates the excitation light from the light to be detected. A flexible main dichroic beam splitter, which operates in the visible range of the spectrum and which is realized using an acousto-optical component, is an acousto-optical beam splitter (AOBS) such as that described in US 2005/046836 A1. For a continuously tunable or broadband excitation from ultraviolet to near-infrared spectral range, this type of AOBS is not suitable. For the mentioned spectral range, at least three different geometries or embodiments are necessary. Besides that, such an AOBS, which is designed for excitation with narrowband laser lines, possesses only a small spectral selection width, and hence has low efficiency particularly in the context of a broadband light source. A flexible adaptation of a continuous spectrum to a method of examination is thus not possible. This disadvantage, namely, that only one or more discrete lines of the spectrum can be selected for the excitation, applies also to the main dichroic beam splitters, which use a polarizing rotation by means of AOTF for the spectral separation.
This disadvantage can be avoided by means of additional components, which enable continuous spectral splitting of the light and at the same time manipulation in this spectrum, definable with respect to the spectral position. Such additional components are proposed, for instance, in DE 19 15 102 and U.S. Pat. No. 6,633,381 B2. The arrangement described there has the disadvantage that three prism spectrometers—one each before and after the dividing element in the excitation beam path, and one in the detection beam path—are necessary. This considerably increases the complexity and the proneness of the arrangement to fail. Further, it is also limited to parallel confocal microscopes with band illumination. Besides that, the variant described in U.S. Pat. No. 6,633,381 B2 has the disadvantage that due to the fixed elements, discrete, predefined wavelength ranges can be separated and detected.
Therefore, the solutions described above do not exploit the full potential of the broadband emitting or variable broadband light sources in the context of their possible applications. The illumination wavelengths, for which, for example, the dyes, with which the specimen is marked, are to be excited to emit, are selectable only to a limited extent. The same applies to the spectral ranges to be detected, in which the solutions available in the prior art are limited solely to the blocks of discrete excitation lines.
In the aforementioned prior art for the coupling of a broadband light source with an LSM as well as for beam separation in an LSM, no further details are provided as to what a flexible method, especially in the context of the imaging, for optimal selection of the illumination and detection wavelengths or bands should look like and no corresponding arrangements are described. One such method is described in US 2005/046836 A1. The illumination and detection wavelengths are thereby selected on the basis of a comparison with a database, based on the characteristics of the dyes with which the specimen is marked. The illumination and the detection spectral ranges are thereby calculated solely on the basis of the individual spectra filed in the databases. As a result, one obtains a setting, which is supposed to be ideal in a purely theoretical sense. However, many object-specific factors, which can have a considerable influence on the results of the measurement, cannot be taken into consideration therein. Included here, for example, is the dependence of the excitation and the emission spectra on the environment of the dye, as has been used in the prior art for some time, as described, for example, in the article of Tsien, Annu. Rev. Neurosci. 12 (1989), page 227 pp. Another example is the appearance of the unknown fluorophores in the object, which can lead—as in the so-called autofluorescence—to undesirable cross excitations.